An individual's genetic background plays an important role in their susceptibility to cancer, disease progression, and response to therapy. Genetic variations can be used as markers to understand complex diseases including cancer, the immune/inflammatory response, and AIDS. The advent of the sequence of the human and other genomes and the ability to type large numbers of genetic markers has led to the need for greatly enhanced computing and analytic methodology. A genome wide association study for breast cancer In collaboration with scientists at the Memorial Sloan Kettering Cancer Institute we completed a whole genome association study of breast cancer in Ashkenazi Jewish subjects with a family history of disease. The scan identified several loci with potentially significant associations. From this data we selected loci for follow up studies in a replication cohort. We were able to confirm that the FGFR2 gene involved in breast cancer in several European American cohorts, is also associated with cancer in Ashkenazi Jewish populations. In addition a locus containing two genes, RNF146 and ECHDC1, was identified positively associated with breast cancer in Ashkenazi subjects. RNF146 was motifs in common with proteins that regulate estrogen receptor. In an assay for estrogen receptor function, RNF146 inhibited estrogen response. To further characterize this protein we expressed the product in bacterial cells. We are collaborating with David Waugh to crystallize the protein to determine its 3-dimensional structure. In addition we are collaborating with Allan Weissman to assess the predicted ubiquitin E3-like ligase activity that the protein is predicted to possess. Complement genes and Age-Related Macular Degeneration By studying variants in the complement B and C2 ( CFH , C2 ) genes we have identified association in this gene in patients with age-related macular degeneration (AMD). AMD is the leading cause of blindness in the elderly and is estimated to effect as many as of 10 million Americans. By examining the genetic variants in the BF and C2 genes significantly protective haplotypes were identified. The complement pathway is important in the response to pathogens causative for cancer. Further study of this important component of the innate immune response could lead to insight into human disease. A deletion that removes the CFHR1 gene adjacent to CFH is highly protective for AMD. We have obtained over 3000 DNAs from subjects in the Age-Related Eye Disease Study, and are typing them for the associated markers to understand the details of the association and identify additional loci. Recently published data implicated the Toll-like receptor 3 (TLR3) gene in AMD. However we have typed this variant in the AREDS cohort, and along with data from 7 other centers failed to replicate this association. Inhibitors of the Hedgehog/Patched Pathway in Cancer Stem Cells The HH/PTCH pathway has been demonstrated to be mutated in virtually all basal cell carcinomas and a portion of medulloblastomas. In addition, many tumors display ligand-dependent activation of the HH/PTCH pathway including pancreas, prostate, gastrointestinal tract, and small cell lung tumors. The steroid-like, natural product molecule cyclopamine specifically binds to SMO, the downstream regulator of the HH/PTCH pathway. Cyclopamine has been shown to inhibit the growth of HH/PTCH-activated cell lines and xenografts. To demonstrate whether cyclopamine inhibits the expression of HH/PTCH target genes we treated prostate (DU-145, LnCaP), gastric (AGS), and breast cancer (SK-BR3) cell lines and confirmed that cyclopamine inhibits proliferation. RNA isolated from treated and untreated DU-145 cells was subjected to quantitative RT-PCR and a reduction in expression of PTCH, SMO, and GLI1, and GLI2 was observed. To further the development of HH/PTCH inhibitors we designed peptides against TM domains and intracellular loops of the SMO protein. SMO is a member of the G-protein-coupled receptor (GPCR) superfamily, and other GPCRs have been targeted by this approach. While peptides against several TM domains failed to have an effect on cell proliferation, peptides against intracellular loops 2 and 3 were potent inhibitors. As with cyclopamine, the active peptides result in a decrease in expression of HH/PTCH target genes. Both cyclopamine and SMO peptides act by eliciting apoptosis, as measured by the TUNEL assay. To move these reagents forward into clinical studies we have performed biodistribution analysis. Radiolabelled peptide was injected into mice and the peptide was found to distribute throughout the body. Efficacy studies are now planned. We have also characterized the methylation of the promoter region of the SMO gene. In cell lines with high expression of SMO, the promoter region is unmethylated; whereas the promoter is methylated in low expressing cells. Analysis of the MSMB protein Our collaborators in DCEG identified variants in the MSMB gene as associated with prostate cancer. This gene encodes microseminoprotein, beta; a protein found in seminal fluid and a known prostate cancer biomarker. One of the associated variants is found in the promoter region of the gene, and affects a predicted CREB transcription factor binding site. Gel shift experiments confirm this prediction. Population history reconstructions using the human FY, LCT and MHC loci as models. The availability of dense single nucleotide polymorphism data permits an understanding of population genetic changes. By studying gene variants in populations we are able to provide insight into the evolutionary forces. Gene frequencies at FY have altered over the past 50,000 years of human evolution as a consequence of exposure to the malarial parasite, Plasmodium vivax. Lactose intolerance attributed to haplotypes at the Lactase-Phlorizin Hydrolase (LCT) gene provides a different insight: Adult persistence of lactose tolerance is a European acquisition arising in the middle-east about 5000 years ago. It is proposed that the genome alterations that provide for lactose tolerance provided an advantage in surviving dehydration. Most people of European ancestry show the effects of this while most Asians and sub-Saharan Africans and native peoples of the Americas and Pacific Islanders Pacific do not. Finally, we have found that there have been major recent shifts in the gene frequencies in the major histocompatibility locus. We have gathered evidence of recent evolutionary change through the study of human population sub-group differences at this locus, and are now beginning to define the specific gene regions within the MHC that may confer differences in individual ability to respond to pathogens. In sum, we believe that population history reconstruction provides a powerful lens on the shaping of our genome, and its liability for heritable human disease.